Oils containing unsaturated fatty acids are used in a variety of food, health and medical applications, such as dietary supplements. Oils containing unsaturated fatty acids can come from a variety of sources, for example, vegetable oils, animal oils and marine oils. Marine oils (e.g. fish oils) comprising omega-3 polyunsaturated fatty acids, in particular, are used in a variety of dietary/therapeutic compositions.
Oils containing unsaturated fatty acids tend to spoil upon storage by oxidation processes. See e.g., Shahidi et al. in BAILEY'S INDUSTRIAL OIL AND FAT PRODUCTS (2005, 6th Ed., Fereidoon Shahidi, ed.), Chapter 8, pages 357-385 . As the oxidation proceeds, various oxidation products are produced that can make the oil unfit for use in health and nutritional applications. When a certain level of these oxidation products is reached, making the oil unfit for use in health and nutritional applications, the oil is spoiled or rancid. Primary oxidation products are hydroperoxides that are relatively unstable and susceptible to further decomposition. Secondary oxidation products thought to be produced from the further reaction of the hydroperoxides include aldehydes, ketones, alcohols, hydrocarbons, volatile organic acids, and epoxy compounds, among others. See id., at 357-359. The oxidation level of oil may be measured by a number of methods, including the measurement of the iodine and peroxide values by titration methods; and the measurement of conjugated dicncs and trienes, 2-thiobarbituric acid value, p-anisidine value, and carbonyl value by spectrophotometric methods. See id.
The p-anisidine value is a common method for measuring secondary oxidation products via a “p-anisidine value” or “anisidine value” using the “p-anisidine test.” See e.g., Shahidi at 368. For example, in evaluating oxidation of salad oils, a study found a high correlation between the anisidine value and flavor scores of the salad oils. See Gray et al., J. Am. Oil Chem. Soc., vol. 55, p. 539-545 (1978). The reaction of p-anisidine with secondary oxidation products, which include unsaturated aldehydes and ketones, produces yellowish products that absorb at 350 nm. See Shahidi at 368. The method is performed using a spectrophotometer, and the anisidine value is given by multiplying the optical density by 100, of a solution of 1.00 gm oil in 100 mL solution containing p-anisidine, at a wavelength of 350 nm. See American Oil Chemists' Society Official Method Cd-18-90. Any materials in solution that absorbs at 350 nm will be included in the anisidine value, because a spectrophotometer does not discriminate between materials that absorb at the same wavelength.
Studies using instrumental methods have been employed to assess the impurities and oxidation level of oils. One such study used solid phase microextraction followed by gas-chromatography-mass spectroscopy to assess fish oils. See Ritter et al., Lipids, vol. 47, p. 1169-1179 (2012). This study required multiple steps and purports to detect over 100 oxidation products in samples of oxidized fish oil. See Ritter at 1173. Another study used high-performance liquid chromatography, gas-chromatography-mass spectroscopy, and 13C NMR to identify oxidation products in fish oil. See Saeed et al., J. Am. Oil Soc. Chem., vol. 76, p. 391-397. This study identified numerous oxidation products in fish oil, and the chromatograms displayed a complex mixture with over-lapping peaks. See Saeed at 394, Fig. 1. Additionally, a study employing Fourier Transform Infrared spectroscopy (FTIR) was used to predict the anisidine value of palm olein. This study used mixtures of oxidized and fresh palm olein to produce a calibration curve for palm olein up to an anisidine value of 17. See Man et al., J Am. Oil Soc. Chem., vol. 76, p. 243-247.
All of the above instrumental methods were able to assess impurities and oxidation of oils with varying success. The chromatographic techniques (see Ritter; see Saeed) produced chromatograms with large numbers of peaks indicating many different compounds, which can make assessment of oxidation of the oil difficult. Additionally, the oxidation impurities from different oils can be different, adding complication to any analysis. The FTIR method (see Man), which according to the authors, worked well for palm olein, would most likely require individual calibration curves to be generated for each type and blend of oil that requires analysis. Thus, a more straightforward and robust method of determining an oxidation level of an oil is needed that does not involve identifying multiple impurities or include multiple calibrations.
The current method for measuring the anisidine value in oils containing unsaturated fatty acids is a simple method but is deficient, at least for the reason that the current anisidine value method produces false positives when certain additives (e.g., flavorings and colorants) are present in the oil. Additives that absorb at 350 nm will contribute to the anisidine value, and produce an anisidine value that is higher than the same oil without the additives present. This overly high value would lead to a measurement that is incorrect, and falsely indicate that the oil oxidized, and thus spoiled or rancid. Therefore, an improved, straightforward method is desired that can more accurately determine the oxidation of oils containing unsaturated fatty acids across a variety of present and future oil formulations and products that contain materials that can interfere with the existing tests.